The Imaging Sciences Laboratory is involved in a major collaborative research effort with the NIH Institutes involving the use of image processing techniques and advanced computational techniques in structural biology to analyze electron micrographs and NMR spectra with the goal of determining macromolecular structures and dynamics. Recent efforts have concentrated on the 3D reconstruction, analysis and interpretation of the structures of icosahedral virus capsids in addition to structure determination and analysis of isolated and complexed proteins and nucleic acids. Ongoing research involves analyses of structures related to papillomavirus and RNA containing virus capsids. In addition we have also been developing computational tools for the study of the structure and dynamics of biological macromolecules using Nuclear Magnetic Resonance (NMR) data. We develop and maintain the Xplor-NIH software package for structure determination, which is used in the NMR labs in the Institutes, and also worldwide. In the past year we have completed the development of an implicit solvent model to use with NMR structure calculations, which allows for much more realistic atom-atom interactions than are usually used in structure determination. We have shown that the use of this new approach can improve the quality of calculated structures, and can allow for accurate structure calculation in the presence of less experimental data. In other work, we have shown that the structure of the HIV capsid protein can be determined in solution using residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. This floppy molecule forms a mixture of monomer and dimer in normal experimental conditions, which can only properly be described using an ensemble of structures. A further complicating characteristic of these structures is that the monomer ensemble is different from the subunit dimer ensemble. Human papillomavirus (HPV) has been implicated as the causative agent in cervical and other epithelial cancers. The major capsid protein (L1), initially forms a loosely connected procapsid which, under in vitro conditions, condenses over several hours into the more familiar 60 nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by 5% in diameter; its pentameric capsomeres change in structure, most markedly in their axial region; and the interaction surfaces between adjacent capsomeres are consolidated. These structural changes are accompanied by the formation of disulfide crosslinks that enhance the stability of the mature capsid. Buffering the lysate to more neutral conditions allowed the production of recombinant capsids with >90% disulfide bonds. The ability to produce fully mature recombinant capsids should benefit further, structural investigations of native HPV capsids. Moreover, fully mature pseudovirions should be viewed as preferred reagents for studies aimed at elucidating the entry pathways used by HPV in the course of natural infections. Current work involves collaborations with a goal to engineer a better HPV vaccine. HPV also contains a minor capsid protein (L2). The L1 sequence is not highly conserved across all types of HPV. L2, on the other hand, is highly conserved. Current (L1 containing) vaccines are roughly 85% effective because they contain a mixture of HPV subtypes. However, since L2 is highly conserved, a vaccine based on L2 could provide close to 100% efficacy. Our goal is to study the structure of a better vaccine candidates (i.e. to try to achieve closer to 100% effectiveness). This is an example of Hi-Risk, Hi-Reward research. Another long-term structural project studies double-stranded RNA containing viruses. This research is ongoing. Proteins take multiple, biologically important conformations in solution. While NMR and small angle scattering data (SAS) are good probes of solution structures, simultaneously determining multiple structures along with their populations is a difficult problem, one which we have been addressing over the past 15 years. We have recently applied these approaches to elucidate very large-scale motion in the Enzyme I protein of the phosphoryl transfer system, important for regulation of sugar uptake by cells. In collaboration with researchers at the University of California, San Diego and the Sanford Burnham Prebys Medical Discovery Institute, we have undertaken the development and application of an implicit solvent model for use in conjunction with experimental data in the structure determination of solution-phase and membrane protein structures. Normally, NMR structure calculations are carried out in the absence of solvent. Including solvent effects in an implicit model has been shown to improve the calculated structures of these types of proteins. We have also revisited the simplified force field used in NMR structure determination of RNA, and replaced and optimized multiple aspects, including parameters for covalent geometry and atomic radii, and modernizing a knowledge-based potential of mean force used to maintain reasonable values of the dihedral angles. The resulting force field greatly assists in improving NMR RNA structures such that they approach the quality of X-ray crystal structures when evaluated using standard validation tools. We have developed and promoted computational methods for incorporating electron microscopy (EM) data into atomic-level structure determination, in concert with NMR data. This work has resulted in two publications. One details the usefulness of EM data in NMR structure determination of NMR. In a second paper NMR data allowed the determination of the conformation of a protein toxin ligand bound to a large ion channel protein involved in heat sensing which had previously been determined using EM.